Effect Of X-rays On Transcription Profiles And Store-operated Ca²⁺ Entry Of Rat Brain Microvascular Endothelial Cells

BackgroundRadiation-induced brain edema is a serious adverse effect of radiotherapy.Although there are many causes of radiation-induced brain edema,the pathogenesis is not clear and clinical treatment is not ideal.Current evidence indicates that brain microvascular endothelial cells（BMECs）are highly sensitive to radiation and may play an important role in this type of edema.BMECs apoptosis causes injuries that include capillary structure abnormalities and blood brain barrier disintegration.Therefore,knowing the differential expression of the BMECs transcriptome after brain radiotherapy may shed light on the pathogenesis of radiation-induced brain edema.Objective1. To explore the changes of transcriptional expression profile of brain microvascular endothelial cells after X-ray irradiation.2.To investigate the effect of X-rays on store-operated Ca²⁺entry of rat brain microvascular endothelial cells.Method1. Primary culture of cerebral microvascular endothelial cellsAfter SD rats were suffocated by CO₂,they were disinfected and brushed with 75%alcohol.The skull was quickly dissected,the dura mater and pia mater were removed,the large blood vessels were removed and the cerebral cortex was collected.The cortex was shredded and homogenized,and quickly filtered through an 80-mesh filter.The filtrate was collected through a 200-mesh sieve.The filter was added with 0.2%type II collagenase,digested at 37°C for 25 minutes,and washed with sterile PBS 3 times to inoculate the cell culture plate,and the solution was changed after 48 hours.Subculture the cells when they reach 80%density.2. X-ray irradiationPrimary cultured cerebral microvascular endothelial cells were divided into three groups,namely the low-dose irradiation group（5Gy）,the high-dose irradiation group（20Gy）,and the control group.A single radiation dose of 5 Gy or 20Gy was delivered to irradiation group,while the control group received the same treatment except that it did not receive X-ray irradiation.3. High-throughput sequencingThe high-dose group and the low-dose group were further divided into two groups at the time points of 12h and 24h after irradiation,and the control group was added to a total of 5 groups,namely:12h after 5Gy irradiation group,24h after 5Gy irradiation group,12h after 20Gy irradiation group,24h after 20Gy irradiation group and control group.Set 3 biological replicates per group.After RNA extraction from these 5 groups of cells,transcripts from 5 groups of brain microvascular endothelial cells were obtained by c DNA library construction and computer sequencing.Compare the four irradiation groups with the control group.The difference factor（FC）is greater than or equal to 1.5,and the P value is less than 0.05.Differentially analyze the transcripts to obtain differentially expressed genes,and perform deep bioinformatics on the differentially expressed genes.analysis.4. Real-time quantitative PCR（q PCR）Using the five groups of brain microvascular endothelial RNA extracted above,the relative quantitative m RNA expression was obtained by reverse transcription and q PCR.5. Calcium imagingSimilarly,the changes in the calcium influx of the primary brain microvascular endothelial cell calcium pool in the above five groups were observed.In the calcium-free buffer solution（0PSS）,the intracellular calcium pool was emptied by adding thapsigargin（TG）,and the difference between the external calcium influx in the control group and the experimental group was observed under the condition that the calcium pool was emptied by adding Ca2+to the buffer solution.Results1.Compared with the control group,40 differentially expressed genes were screened in the irradiation group:Zfp263,Zfp217,Tp53Inp1,Tfap2B,Tap1,Svop,Sgk1,Rf00560,Rf00026,Rac1,Rab40C,Puf60,Psrc1,Plk2,Pla2G16,Orai2,Nr4A1,Lpl,Loc100912596,Lbh,Klhl20,Kin,Hmox1,Hint3,Glrx3,Gdf6,Gdf15,Fzd7,Fos,Fas,Egr2,Edn1,Cyp2T1,Cebpd,Ccdc117,Calhm2,Bok,Atp5Ac.These differentially expressed genes are involved in biological processes such as cell cycle,apoptosis,vascular permeability,and calcium signal regulation.2.Among the 40 differentially expressed genes,the expression level of Orai2 in the irradiation group was significantly higher than that in the control group,and the expression level increased over time.3.Real-time quantitative PCR showed that the expression level of Orai2 in the 5Gy and20Gy irradiation groups was significantly higher than that in the control group,and the expression level gradually increased over time,which was consistent with the sequencing results.4.On the premise of depletion of the intracellular calcium bank of the brain microvascular endothelial cells,the extracellular calcium infusion was significantly stronger than that in the control group.Conclusion1.The expression of Orai2 in brain microvascular endothelial cells after X-ray irradiation was significantly increased,and the up-regulation of Orai2 may be an important cause of radioactive brain edema.2.SOCE was enhanced in the brain microvascular endothelial cells after irradiation,which may be one of the mechanisms that induce radioactive brain edema...